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Interactions of polysaccharides with β-lactoglobulin spread monolayers at the air–water interface
Food Hydrocolloids 18 (2004) 959–966 www.elsevier.com/locate/foodhyd
Interactions of polysaccharides with b-lactoglobulin spread monolayers at the air – water interface R. Baezaa, C. Carrera Sanchezb, A.M.R. Pilosofa, J.M. Rodrı´guez Patinob,* a
Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria (1428), Buenos Aires, Argentina b Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, c/. Prof. Garcı´a Gonza´lez, 1. 41012, Sevilla, Spain Received 5 November 2003; revised 26 January 2004; accepted 5 March 2004
Abstract In the present work we have studied the static (film structure and elasticity) and dynamic characteristics (surface dilatational properties) of b-lactoglobulin (bLG) monolayers spread at the air –water interface in the presence of polysaccharides in the aqueous phase, at 20 8C and at pH 7. The measurements were performed on a fully automated Wilhelmy-type film balance. As polysaccharides with interfacial activity we have used propylene glycol alginates (PGA). To evaluate the effect of the degree of PGA esterification and viscosity, different commercial samples were studied-kelcoloid O (KO), kelcoloid LVF (KLVF) and manucol ester (MAN). Xanthan gum (XG) and l-carrageenan (lC) were studied as non-surface active polysaccharides. The results reveal a significant effect of surface active and non-surface active polysaccharides on static—when the polysaccharide was added in the subphase the p-A isotherms shifted to higher surface pressure values as the time increased-and dynamic—the presence of polysaccharide in the aqueous phase decreased the surface dilatational modulus of a pure b-lactoglobulin monolayer-characteristics of b-lactoglobulin monolayers. To explain the observed effects three phenomena were taken into account: (i) the ability of the polysaccharide to adsorb at the interface by it-self and to increase the surface pressure, (ii) the interfacial complexation of the polysaccharide with the adsorbed protein and (iii) the existence of a limited thermodynamic compatibility between the protein and polysaccharide, depending on the protein-polysaccharide system. q 2004 Elsevier Ltd. All rights reserved. Keywords: Protein; Polysaccharide; Gums; Hydrocolloid; Air–water interface; Monolayer; Surface tension; Interfacial rheology
1. Introduction Proteins and polysaccharides are present in many kinds of foods (Dickinson, 1992). The main role of proteins in foamed products is to stabilise the air –water interface through their capacity to lower the surface tension of water (Damodaran & Paraf, 1997). Polysaccharides are widely used for stabilisation of food emulsions and foams (Stephen, 1995; Renard, Della Valle, & Popineau, 2002). Most highmolecular-weight polysaccharides, being hydrophilic, do not have much of a tendency to adsorb at the air– water interface, but they can strongly enhance the stability of protein foams by acting as thickening or gelling agents (Dickinson, 2003). Alongside the use of non-surface-active polysaccharides in food foams as thickeners, there is some evidence that supports an additional role with some polysaccharides at the interfacial film. The formation of * Corresponding author. Tel.: þ 349-5-4556446; fax: þ349-5-455-7134. E-mail address: [email protected] (J.M. Rodrı´guez Patino). 0268-005X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2004.03.006
protein – polysaccharide complexes has been related to enhance the functionality of proteins adsorbed at fluid interfaces (Dickinson, 2003). Protein – polysaccharide interactions are sensitive to details of protein and polysaccharide structures as well as to pH (Dickinson, 2003; Tolstoguzov, 1997). Aqueous solutions of proteins and polysaccharides can exhibit one of the following phenomena: complex coacervation (complexation), miscibility and segregation. Complex coacervation occurs below the protein isoelectric point as a result of net electrostatic interactions between the biopolymers carrying opposite charges and implies the separation of two phases, one rich in the complexed biopolymers and the other phase depleted in both. Above the isoelectric point of the protein thermodynamic incompatibility between the protein and polysaccharide generally occurs because of the repulsive electrostatic interactions and different affinities towards the solvent (Tolstoguzov, 1997). Therefore, protein and polysaccharide
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may co-exist in a single phase (miscibility) in domains in which they mutually exclude one another or, above a critical concentration, segregate into different phases. Excluded volume effects can have the following manifestations: enhancement of the association of macromolecules, reduction in the critical concentration for gelation and increase in the rate of gelation (Capron, Nicolai, & Durand, 1999; Baeza, & Pilosof, 2001), enhancement of protein adsorption at fluid interfaces (Tsapkina, Semenova, Pavlovskaya, & Tolstoguzov, 1992). In the present work we have studied the static and dynamic characteristics of b-lactoglobulin (bLG) monolayers spread at the air –water interface in the presence of polysaccharides in the aqueous phase, at 20 8C and at pH 7. As polysaccharides with interfacial activity we have used propylene glycol alginates (PGA). To evaluate the effect of the degree of PGA esterification and viscosity, different commercial samples were studied. Xanthan gum (XG) and l-carrageenan (lC) were studied as non-surface active polysaccharide. Sulphated polysaccharides like carrageenans can interact with the positively charged groups in a protein more strongly than carboxylated hydrocolloids like XG at pH above the protein isoelectric point (Dickinson, 2003). Rheological properties of XG, along with its pH and temperature stability make it an ideal thickener for food emulsions and foams. XG is an anionic polysaccharide that produces high viscosities at low concentrations (Imeson, 1992). Enhanced hydration is attributed to side chains of anionic character consisting in units of a-D mannnose, b-D glucuronic and pyruvate, occurring on the cellulose structure backbone of xanthan. Xanthan, being highly hydrophilic and without any significant hydrophobic bonds, is not adsorbed at the air– water interface (Yilmazer, Carrillo, & Kokini, 1991). Nevertheless, xanthan promoted soy protein subunits aggregation at the air –water interface in foams based on native soy proteins (Carp, Bartholomai, & Pilosof, 1999). Those specific effects further influenced the foam stability of the mixed systems (Carp, Bartholomai, Relkin, & Pilosof, 2001). One possible interpretation of these findings is that the gum adsorbs onto the protein, forming a combined structure of a primary protein layer predominantly in contact with the air phase. Following this last hypothesis, the surface tension might be dominated by the primary protein layer, covered by and adsorbed polysaccharide layer in strong electrostatic contact, dominating the surface rheology (Galazka & Dickinson, 1995). One distinctly group of surface-active polysaccharides are the propylene glycol esters of alginic acid (PGA), a high molecular weight linear polysaccharides composed of 1,4 linked- D -mannuronic acid and L -guluronic acid (Sarker & Wilde, 1999). There are produced with a range of viscosities and degrees of esterification. The increasing of the degree of esterification reduces the overall hydrophilic character of the molecules and imparts surface-active properties. The resulting ability of PGA to reduce the surface tension of
water as well as increase the viscosity of the water phase make them suitable as stabilisers and foaming agents. Substitution of more than 50% of the carboxyl groups also produces greater acid and calcium ion tolerance than other alginates. Propylene glycol alginate is an example of a polysaccharide used in food foams, such as beer, to aid ‘head’ retention of the foam (Sarker & Wilde, 1999). Due to its surface-active character, competitive adsorption of PGA could occur in mixtures of this polysaccharide and proteins. In addition, formation of protein –polysaccharide complexes at the interface could also occur. The interaction between PGA and milk proteins in foams was studied by Ahmed and Dickinson (1991). The foaming properties were strengthened at pH value above 7, where both biopolymers carried negative charges, revealing the possibility of formation of electrostatic complexes between positively charged patches in the proteins and carboxyl groups in PGA. A more quantitative indication of non-covalent complex formation between PGA and caseinate, whey protein isolates and a-lactalbumin at the oil– water interface was provided by film surface viscosity (Dickinson & Euston, 1991). More recently, the foam stability of a mixed bovine serum albumin/Tween 20 solution at pH 4.2 was found to be significantly increased in the presence of PGA (Sarker & Wilde, 1999). The principal mechanism is thought to be non-covalent cross-linking of the protein molecules at the surface. b-lactoglobulin (bLG), the most abundant protein in whey, is a globular protein of molecular mass 18.3 kDa, stabilised by two internal disulfide cross-linking, that exhibits good foaming properties (Tornberg, 1979). The structural and dynamic properties of b-lactoglobulin at the air –water interface, have been extensively studied in the past years (Horne & Rodrı´guez Patino, 2003; Rodrı´guez Nin˜o, Carrera, Cejudo, & Rodrı´guez Patino, 2001, Rodrı´guez Patino, Carrera, Rodrı´guez Nin˜o, & Cejudo, 2001) so that this protein is a good model to study the interactions of non-surface active and surface active polysaccharides with proteins at the air –water interface.
2. Materials and methods 2.1. Materials b-lactoglobulin (bLG) was supplied by Danisco Ingredients (Denmark). The powder composition was: protein 92%, b-lactoglobulin . 95%, a-lactalbumin , 5%. The polysaccharides l-carrageenan (lC) and xantham gum (XG) were provided by BIOTEC (Argentina) and propylene glycol alginates (PGA) were from ISP Alginates Inc. (USA). The PGA used were: kelcoloid O (KO), kelcoloid LVF (KLVF) and manucol ester (MAN). The degree of esterification and viscosity of propylene glycol alginates are shown in Table 1. The surface pressure of aqueous solutions of polysaccharides at 0.1 and 0.5 wt%—which
R. Baeza et al. / Food Hydrocolloids 18 (2004) 959–966 Table 1 Degree of esterification and viscosity of propylene glycol alginates PGA
Degree of esterification
Viscositya
Manucol ester (MAN) Kelcoloid LVF (KLVF) Kelcoloid O (KO)
High Medium High
High (11.8 cps) High (13.9 cps) Low (4.7 cps)
a
viscosity (60 s21) of 0.5% wt/wt solution.
was determined using an automatic drop tensiometer as described elsewhere (Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 1999)—are shown in Table 2. It can be seen that all the PGA used in this study have a significant surface activity denoting the surface-active character of these polysaccharides. The surface pressure of XG is practically zero denoting the non-surface active characteristic of this polysaccharide. However, the surface pressure of l-carrageenan is different to zero denoting the presence of some impurities in the commercial sample. Thus, a purification of this polysaccharide was necessary before the experiments in the surface film balance, by the continuous suction of the adsorbed impurities until the surface pressure of the aqueous solution turns to zero.
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complete spreading, adsorption and rearrangements of the protein, 30 min was allowed to elapse before measurements were taken. In the systems with surface-active polysaccharides in the aqueous phase the protein was spread at the interface using pure water as sub-phase. Then a concentrated PGA solution was added to the sub-phase with a syringe. The final concentration of polysaccharide in the sub-phase was approximately 0.05% wt/wt in. In the presence of nonsurface active polysaccharides (lC and XG), the protein was spread directly on the surface of polysaccharide solutions at 0.05% wt/wt. The sub-phase temperature was maintained at 20 8C by water circulation from a thermostat, within an error range of 0.5 8C. The reproducibility of the surface pressure results was better than ^ 0.4 mN/m. 2.3. Film elasticity The elasticity was calculated from the slope of the p-A isotherm as E ¼ 2Aðdp=dAÞT : The elasticity defined here is the elasticity at zero deformation rate. The film elasticity is a measure of the film resistance to a change in area and can be calculated directly from the slope of the p-A isotherm at constant temperature.
2.2. Surface film balance
2.4. Surface dilatational rheology
The measurements of the surface pressure ðpÞ vs. average area per molecule (A) were performed on fully automated Wilhelmy-type film balance (KSV 3000, Finland) as described elsewhere (Rodriguez Patino et al., 2001). The maximum area of the trough between the two oscillating barriers is 51.5 £ 15 cm2. To form the protein surface film, bLG was spread in the form of a solution, using Milli-Q ultrapure water at pH 7, adjusted by a commercial buffer solution (Sigma, . 99.5%) called trizma((CH2OH)3CNH2/ (CH2OH)3CNH3Cl). Ionic strength was 0.05 M in all the experiments. The sub-phase solutions consisted in 0.05% wt/ wt polysaccharide solutions prepared with Milli-Q water. Aliquots of 400 ml of aqueous solutions of bLG (1.6 1024 mg/ml) at pH 7 were spread on the interface using the method developed by Trurnit (1960). To allow
Determination of surface rheological parameters—such as surface dilatational modulus ðEÞ; its elastic and viscous components, and the loss angle tangent—were carried out in a home modified-Wilhelmy-type film balance as described elsewhere (Rodriguez Patino et al., 2001). In this method the surface was subjected to small periodic sinusoidal compression and expansion cycles by means of two oscillating barriers at a given frequency ðvÞ and amplitude ðDA=AÞ and the response of the surface pressure ðpÞ was monitored. The conditions used in this study were a frequency of 50 mHz and an amplitude of 5%. Surface pressure was directly measured by means of two roughened platinum plates situated on the surface between the two barriers. The surface dilatational modulus ðEÞ derived from the change in surface pressure resulting from a small change in surface area may be described (Lucassen and van den Tempel, 1972) by E ¼ 2dp=d ln A: The dilatational modulus is a complex quantity, composed of real (the surface dilatational elasticity, Ed ¼ lEl cos u) and imaginary (the surface dilatational viscosity, Ev ¼ lEl sin uÞ parts. The loss angle tangent tanu ¼ Ev =Ed is a measurement of the solid character of the film and is near zero for pure elastic films.
Table 2 Surface pressure of aqueous solutions of polysaccharides at 20 8C and at pH 7, after 3600 s of adsorption Concentration of polysaccharide in the aqueous solution
Manucol ester (MAN) Kelcoloid LVF (KLVF) Kelcoloid O (KO) Xanthan gum (XG) l-Carrageenan (lC)
0.1%wt
0.5%wt
6.8 6.3 9.3 <0 –
6.9 6.3 9.3 <0 7.8a
a The surface pressure of a l-Carrageenan solution at 0.5%wt was zero after the suction of the surface-active impurity.
3. Results 3.1. Monolayer structural characteristics The monolayer structural characteristics can be obtained through the p-A isotherm. The p-A isotherm for
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b-lactoglobulin spread on aqueous subphase at pH 7 is shown in Fig. 1. b-lactoglobulin monolayers have a liquidexpanded-like structure (Rodriguez Patino et al., 2001). It appears that a critical surface pressure and surface concentration exist at which the film properties change significantly. This critical surface pressure of 12 mN/m represents the transition between a monolayer with a more expanded structure (state 1) towards a monolayer in which the proteins form a more condensed structure (state 2). Nevertheless, according to Graham and Phillips (1979), b-lactoglobulin would retain elements of the native structure not fully unfolded at the interface, even in the more expanded configuration (Rodriguez Patino et al., 2001). When the surface active PGA were added in the subphase at a concentration of 0.05% wt/wt, the p-A isotherms shifted to higher surface pressure values as the time increased, due to the adsorption and penetration of the polysaccharide into the protein monolayer (Figs. 1– 3). The reproducibility of the isotherms after continuous compression –expansion cycles was reached after 18 – 22 h, indicating that the adsorption and penetration of PGA into the protein monolayer and its subsequent rearrangement is a slow process. In the presence of non-surface active polysaccharides different results were obtained. The presence of XG strongly shifted the p-A isotherms to higher surface pressure values as the time increased (Fig. 4), but l-carrageenan induced only small changes in the p-A isotherm (data not shown). The p-A isotherms at the maximum adsorption time (the pseudo steady state p-A isotherm) of b-lactoglobulin and b-lactoglobulin þ polysaccharide mixed systems are shown in Fig. 5. It can be seen that the p-A isotherms for b-lactoglobulin þ PGA and b-lactoglobulin þ XG were separated from the surface pressure axis indicating an apparent less condensed monolayer structure because in the molecular area we assume that only bLG molecules are
Fig. 1. p-A isotherms of b-lactoglobulin monolayers spread on aqueous solutions of Kelcoloid O (KO) at 20 8C and at pH 7. Key: (1, —) blactoglobulin spread on pure water and after the addition of Kelcoloid O to the aqueous phase and after a waiting time of (2, pppp) 16 h and (3, ---) 18 h.
Fig. 2. p-A isotherms of b-lactoglobulin monolayers spread on aqueous solutions of Kelcoloid LVF (KLVF) at 20 8C and at pH 7. Key: (1, —) b-lactoglobulin spread on pure water and after the addition of Kelcoloid LVF to the aqueous phase and after a waiting time of (2, – –) 4 h, (3, · · ·) 18 h, and (4, -·-) 22 h.
spread on the interface. Among the different PGA, MAN showed the highest surface activity as it induced the highest surface pressure increase. KLVF and KO induced a much lower increase in surface pressure. Differences among them were only apparent at low molecular areas where KLVF exhibited slightly higher surface activity. In spite of the lack of a surface activity by it-self, xanthan showed a behaviour similar to PGA. Moreover, at low molecular areas XG induced higher surface pressure than KLVF and KO. The l-carrageenan slightly increased the surface pressure at low molecular areas. By plotting the elasticity versus surface pressure for pure bLG and mixed systems (Fig. 6) a more clear evidence of the changes in the monolayer structure may be obtained. The elasticity of pure bLG monolayer increased with the surface pressure, reached a maximum as the change
Fig. 3. p-A isotherms of b-lactoglobulin monolayers spread on aqueous solutions of Manucol ester (MAN) at 20 8C and at pH 7. Key: (1, —) b-lactoglobulin spread on pure water and after the addition of MAN to the aqueous phase and after a waiting time of (2, – –) 1 h, (3, · · ·) 18 h, and (4, -·-) 22 h.
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Fig. 4. p-A isotherms of b-lactoglobulin monolayers spread on aqueous solutions of xanthan gum (XG) at 20 8C and at pH 7. Key: (1, —) b-lactoglobulin spread on pure water and after the addition of xanthan gum to the aqueous phase and after a waiting time of (2, – –) 1.5 h, (3, · · ·) 13 h, and (4, -·-) 14 h.
between states 1 and 2 takes place and then decreased at the higher surface pressures as the monolayer collapses and the formation of a multilayer takes place (Rodriguez Patino et al., 2001). Fig. 6 shows that the influence of the polysaccharide prevails at low surface pressures (at p , 12 mN/m) as bLG adopts the state less condensed (state 1). In this region the elasticity of the film was reduced up to 50% by the presence of MAN, KO and KLVF (Fig. 6a). This effect may be due to the penetration of polysaccharides in the protein film, which hinder the interactions within the protein aminoacid residues at the air –water interface. The non-surface active XG and lC also reduced the elasticity of pure bLG monolayer (Fig. 6b) but in a lower extent than surface active polysaccharides. This effect may be due to the interactions between protein and polysaccharide that affect the original structure of the protein monolayer. These results
Fig. 6. Surface pressure dependence of the elasticity of (—) b-lactoglobulin monolayers spread on water and after the addition of polysaccharides to the aqueous phase. (a) Surface active polysaccharide: (K) Kelcoloid O, (q) Kelkoid KLF, and (W) Manucol ester. (b) Non-surface active polysaccharide: (W) l-carrageenan, and (S) xanthan gum. Temperature 20 8C. pH 7.
would also suggest that the protein in the presence of these polysaccharides would be forced to adopt the more condensed state (state 2) even at low surface pressures. In the region where the protein adopts the state 2 (at 12 , p , 25 mN/m), the elasticity decreased and lower differences were observed among the systems (the elasticity varied within 20 and 30 mN/m). Above a surface pressure of 25 mN/m, in the monolayer collapse region, the elasticity tended to decrease no matter what the polysaccharide. 3.2. Surface dilatational characteristics
Fig. 5. Pseudo steady state p-A isotherms of b-lactoglobulin monolayers spread on aqueous solutions of polysaccharides at 20 8C and at pH 7. Key: (1, —) b-lactoglobulin spread on pure water and after the addition of (2, ----) Kelcoloid O, (3, · · ·) Kelkoid KLF, (4, -·-) Manucol ester, (5, -··-) l-carrageenan, and (6,þþ þ ) xanthan gum to the aqueous phase.
The values of surface dilatational modulus (data not shown) were very similar to that of the dilatational elasticity supporting the elastic character of the film. The dilatational elasticity ðEd Þ of bLG monolayer behaved as previously reported (Rodriguez Patino et al., 2001). The dilatational elasticity increased with surface pressure up to a maximum close to the surface pressure at which the monolayer collapses (Fig. 7). The presence of lC in the aqueous
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Fig. 7. Surface pressure dependence of the surface dilatational elasticity (Ed) of (A) b-lactoglobulin monolayers spread on water and after the addition of polysaccharides to the aqueous phase. Surface active polysaccharide: (K) Kelcoloid O, (L) Kelkoid KLF, and (W) Manucol ester. Non-surface active polysaccharide: (X) l-carrageenan, and (V) xanthan gum. Temperature 20 8C. pH 7.
subphase slightly modified the dilatational elasticity of the monolayer. A small decrease in Ed was observed at low surface pressure and at the collapse point. The PGA strongly decreased Ed at all surface pressures, being MAN the PGA that more decreased the solid character of the film, especially at the higher surface pressures. XG showed a very strong effect as it produced the largest reduction in Ed ; mainly at surface concentrations below 20 mN/m. b-Lactoglobulin monolayer behave, from a rheological point of view, as viscoelastic, with a loss angle tangent ðtan uÞ within 0.05 –0.2 (Fig. 8). These low values point out the like-gel character of the monolayers (Tung & Dynes, 1982). tan u decreased with increasing surface pressure in agreement with the existence of higher protein– protein
Fig. 8. Surface pressure dependence of the loss angle tangent (tan u) of (A) b-lactoglobulin monolayers spread on water and after the addition of polysaccharides to the aqueous phase. Surface active polysaccharide: (K) Kelcoloid O, (L) Kelkoid KLF, and (O) Manucol ester. Non-surface active polysaccharide: (X) l-carrageenan, and (V) xanthan gum. Temperature 20 8C. pH 7.
interactions. Except XG at lower surface pressures, the presence of polysaccharide decreased the viscoelasticity of bLG monolayer. Among PGA, KO less affected the viscoelastic character of the monolayer and MAN and KLVF showed a similar behaviour. The non-surface active polysaccharides showed opposite effects on the viscoelastic character of the film. At low surface pressures (in the region where the protein adopts the state 1), lC strongly decreased the viscoelasticity but the opposite was observed with XG. In fact, in the presence of lC the viscous component of the dilatational modulus of the mixed system increased in comparison to the protein alone. However, the presence of xanthan greatly decreased the viscous component of the dilatational modulus resulting in the increase of the viscoelastic character of the film.
4. Discussion The results of static (film structure) and dynamic (surface dilatational properties) of b-lactoglobulin and polysaccharide mixed systems reveal a significant effect of polysaccharide, even of the non-surface active xanthan and lC, on bLG monolayer. To explain the observed effects three phenomena must be taken into account: (i) the ability of the polysaccharide to adsorb at the interface by itself and to increase the surface pressure, (ii) the interfacial complexation of the polysaccharide with the adsorbed protein and (iii) the existence of a limited thermodynamic compatibility between the protein and polysaccharide. Anchorage of the polysaccharide at the interfacial film may occur by mechanism (i) or (ii), depending on the chemical structure of the polysaccharide and on the pH. Once the polysaccharide is into or attached by complexation to the interface, exclusion volume effects between both biopolymers at neutral pH could lead to a rise of chemical potential or, in other words, to a modification of the thermodynamic activity of the protein at the interface (Pavlovskaya, Semenova, Thzapkina, & Tolstoguzov, 1993). Therefore, the protein at the air – water interface would perform as a more concentrate film (i.e. with a more condensed structure), leading to an increase in the surface pressure, according to the results of Figs. 1– 3 and 5. It has been demonstrated that xanthan addition to soy protein solutions at neutral pH had an effect similar to that observed by increasing protein concentration arising mainly from excluded volume effects (Carp et al., 1999). The implication of some protein patches in electrostatic interactions with XG or lC or the penetration of PGA into the interface would interfere with the interactions between bLG molecules resulting in a decreasing in the surface dilatational elasticity (Fig. 7). The decreased elasticity at p , 12 mN/m, as bLG adopts the state 1 (Fig. 6), and the decreased surface dilatational elasticity at every surface pressures (Fig. 7) of bLG monolayers in the presence of polysaccharide reveals a decreasing in the degree of protein association at
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the interface, as well as the increased fluidification of the film (as observed by the increased tan u in Fig. 8, which is consistent with the existence of lower protein– protein interactions in the presence of polysaccharides. b-Lactoglobulin spread films on aqueous solutions of non-surface active polysaccharides. The adsorption of pure xanthan (XG) and l-carrageenan (lG) at the air– water interface is unlikely. Xanthan did not cause a decrease in surface tension (Carp et al., 2001), which was consistent with previous reports (Yilmazer et al., 1991) and purified lC behaved similarly (Table 2). Thus, the modification of surface pressure (Figs. 4 and 5) and rheological properties (Figs. 6b, 7, and 8) of the protein monolayer in the presence of non-surface active polysaccharide in the aqueous phase necessary suggests the participation of XG and lC at the interface by the mechanism (ii). At the neutral pH used in this study, a protein – polysaccharide repulsive interaction may be expected. b-Lactoglobulin is negatively charged and XG is a nonionic polysaccharide with side chains of anionic character consisting in units of a-D mannose, b-D glucuronic and pyruvate, occurring on the cellulose structure backbone. On the other hand, l-carrageenan is a highly sulphated polysaccharide, negatively charged at neutral pH. In bulk solution, the mixtures of whey proteins and XG or lC at pH 7 appeared to be governed by segregative or limited thermodynamic compatibility phenomena (Sanchez, Schmitt, Babak, & Hardy, 1997; Baeza & Pilosof, 2001). However, local net attractive interactions between proteins and polysaccharides may also occur. Under the adsorption of the protein at the interface the character of protein – polysaccharide interactions may be different than in bulk solution because of the altered conformation of protein at the interface. One of the most important properties that differentiate carrageenan from other hydrocolloids is its ability to complex or interact with milk proteins. Sulphated polysaccharides form fairly strong reversible complexes with proteins, even at neutral pH (Dickinson, 2003). Interestingly, lC is the polysaccharide that has a lower effect on bLG monolayer structure (Fig. 5) and surface dilatational elasticity (Fig. 7), but conversely produces a higher fluidification in the film (Fig. 8). Clearly, the interfacial complexation of polysaccharide with adsorbed protein and/ or the repulsive interactions between both biopolymers, according to mechanism (ii), may produce complex effects on the interfacial characteristics (both static and dynamic) of the mixed films. Evidence for the synergistic interaction of xanthan and soy proteins at the air– water interface was previously provided by measurements of surface shear viscosity (Carp, Elizalde, Bartholomai, & Pilosof, 1997) and electrophoresis analysis of protein aggregation at the air – water interface (Carp et al., 1999). One possible interpretation of these findings is that the gum adsorbs onto the protein, forming a combined structure with a primary protein layer
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predominantly in contact with the air phase. Following this last hypothesis, the surface pressure might be dominated by the primary protein layer and should not be changed by the polysaccharide that would mainly dominates the surface rheology (Fig. 7). However, the strong increase of surface pressure observed in bLG monolayer in the presence of xanthan (Figs. 4 and 5) may be taken as an evidence of the ‘concentrating effect’ (condensation of the bLG monolayer structure) arising from the limited thermodynamic compatibility between the biopolymers, according to the mechanism (iii). b-Lactoglobulin spread films on aqueous solutions of surface active polysaccharides. As a result of the ester group, propylene glycol alginates (PGA) reduce the surface tension of water, a phenomenon, which imparts a surface active character to these polysaccharides. The penetration of PGA at the interface, according to mechanism (i) may reduce the protein– protein interactions, which explains the lower values of the surface dilatational elasticity (Fig. 7). An increasing in the degree of esterification reduces the overall hydrophilic character of the molecule and imparts higher surface active properties to the polysaccharide aqueous solution. PGA also differ in their viscosities, and thus in their molecular weights (Imeson, 1992). The surface activity of PGA (in the absence of the protein) followed the order KO . MAN . KLVF (Table 2). The highest surface activity of KO is consistent with the high degree of esterification and low viscosity (i.e. with the lower molecular weight) of this PGA. On the other hand, KLVF because of the lowest degree of esterification and higher viscosity showed the worst performance. Nevertheless, when PGA is adsorbed onto the bLG film, the order of effectiveness in increasing surface pressure (Figs. 1– 3 and 5) was MAN . KLVF . KO. This result cannot be only attributed to the high degree of esterification and necessarily points out the role of the viscosity (i.e. the molecular weight) of PGA, which is indicative of the degree of incompatibility with the protein. The increasing of the molecular weight of the PGA is expected to reduce the biopolymer compatibility (Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998). According to this, the different effects of PGA on p-A isotherms (Fig. 5) may be explained as follows. The greatest increase of surface pressure caused by MAN (Figs. 3 and 5) should be attributed to its high degree of esterification, which allows it to penetrate into the interface. Nevertheless, the possibility of interfacial complexing must not be discarded. Ahmed and Dickinson (1991) suggested the possibility of formation of electrostatic complexes between PGA and milk proteins in foams at pH 7. In addition, due to its high viscosity/molecular weight they would force the protein to concentrate at the interface due to exclusion volume effects. Even if KO had a degree of esterification similar to MAN, due to its low viscosity, would contribute to a lesser extent to concentrate the protein at the interface.
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Finally, the fact that all the polysaccharides, being surface-active or not, caused the transition towards state 2 to occur at lower surface areas is an evidence of the existence of a volume exclusion effects at the interface. Otherwise, the non-surface active polysaccharides would only modify the rheology of the protein film without affecting the surface pressure.
Acknowledgements This research was supported by CYTED through project XI.17 and CICYT through grant AGL2001-3843-C02-01. The authors also acknowledge the support from Universidad de Buenos Aires and Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas de la Repu´blica Argentina.
References Ahmed, M., & Dickinson, E. (1991). Foaming solutions of protein þ propylene glycol alginate. Food Hydrocolloids, 91, 395–402. Baeza, R., & Pilosof, A. M. R. (2001). Mixed biopolymer gel systems of blactoglobulin and nongelling gums. In E. Dickinson, & R. Miller (Eds.), Food Colloids 2000: Fundamentals and Formulation (pp. 394 –403). Cambridge: The Royal Society of Chemistry. Capron, I., Nicolai, T., & Durand, D. (1999). Heat induced aggregation and gelation of b-lactoglobulin in the presence of k-carrageenan. Food Hydrocolloids, 13, 1–5. Carp, D. J., Elizalde, B. E., Bartholomai, G. B., & Pilosof, A. M. R. (1997). Foaming properties of soy proteins as affected by xanthan gum. In R. Jowitt (Ed.), Engineering and Food (pp. 69–72). UK: Academic Press. Carp, D. J., Bartholomai, G. B., & Pilosof, A. M. R. (1999). Electrophoresis studies for determining soy proteins–xanthan gum interactions in foams. Colloids and Surfaces B: Biointerfaces, 12, 309–316. Carp, D. J., Bartholomai, G. B., Relkin, P., & Pilosof, A. M. R. (2001). Effects of denaturation on soy protein–xanthan interactions: comparison of a whipping-rheological and a bubbling method. Colloids and Surfaces B: Biointerfaces, 21, 163–171. Damodaran, S., & Paraf, A. (1997). Food proteins and their applications. New York: Marcel Dekker. Dickinson, E. (1992). An Introduction to Food Colloids. Oxford: Oxford University. Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids, 17, 25– 40. Dickinson, E., & Euston, S. R. (1991). Stability of emulsions containing both protein and polysaccharide. In E. Dickinson (Ed.), Food Polymers, Gels and Colloids (pp. 132– 146). Galazka, V. B., & Dickinson, E. (1995). Surface properties of protein layers adsorbed from mixtures of gelatin with various caseins. Journal of Texture Studies, 26, 401– 409. Graham, D. E., & Phillips, M. C. (1979). Proteins at liquid interfaces. III. Molecular structures of adsorbed films. Journal of Colloid Interface Science, 70, 427 –433.
Horne, D. S., & Rodrı´guez Patino, J. M. (2003). Adsorbed biopolymers: behavior in food applications. In M. Malmsten (Ed.), Biopolymers at Interfaces (pp. 857–900). New York: Marcel Dekker. Imeson, A. (1992). Thickening and gelling agents for foods. New York: Blackie Academic and Professional. Lucassen, J., & van den Tempel, M. (1972). Dynamic measurements of dilatational properties of a liquid interface. Chemical Engineering Science, 27, 1283–1291. Pavlovskaya, G. E., Semenova, M. G., Thzapkina, E. N., & Tolstoguzov, V. B. (1993). The influence of dextran on the interfacial pressure of adsorbing layers of 11S globulin vicia faba at the planar n-decane/ aqueous solution interface. Food Hydrocolloids, 7, 1 –10. Renard, D. (2002). In D. Renard, G. Della Valle, & Y. Popineau (Eds.), Plant biopolymer science. Food and non-food applications. Cambridge: The Royal Society of Chemistry. Rodrı´guez Nin˜o, M. R., Carrera, C., Cejudo, M., & Rodrı´guez Patino, J. M. (2001). Protein and lipid films at equilibrium at air–water interface. Journal of American Oil Chemist’s Society, 78, 873–879. Rodrı´guez Patino, J. M., Rodrı´guez Nin˜o, M. R., & Carrera Sa´nchez, C. (1999). Adsorption of whey protein isolate at the oil–water interface as a function of processing conditions: a rheokinetic study. Journal of Agriculture and Food Chemistry, 47, 3640–3648. Rodrı´guez Patino, J. M., Carrera, C., Rodrı´guez Nin˜o, M. R., & Cejudo, M. (2001). Structural and dynamic properties of milk proteins spread at the air – water interface. Journal of Colloid Interface Science, 242, 141 –151. Sanchez, C., Schmitt, C., Babak, V. G., & Hardy, J. (1997). Rheology of whey protein isolate xanthan mixed solutions and gels. Effect of pH and xanthan concentration. Nahrung, 41, 336 –343. Sarker, D. K., & Wilde, P. J. (1999). Restoration of protein foam stability through electrostatic propylene glycol alginate-mediated protein – protein interactions. Colloids and Surfaces B: Biointerfaces, 15, 203 –213. Schmitt, C., Sanchez, C., Desobry-Banon, S., & Hardy, J. (1998). Structure and technofunctional properties of protein-polysaccharide complex: a review. Critical Review Food Science and Nutrition, 38, 689–753. Stephen, A. M. (1995). Food polysaccharides and their applications. New York: Marcel Dekker. Tolstoguzov, V. B. (1997). Protein–polysaccharide interactions. In S. Damodaran, & A. Paraf (Eds.), Food proteins and their application (pp. 171 –198). New York: Marcel Decker. Tornberg, E. (1979). The absorption behavior of proteins at an interface as related to their emulsifying properties. Journal Science Food Agriculture, 29, 762 –773. Trurnit, H. J. (1960). A theory and method for the spreading of protein monolayers. Journal Colloid Science, 15, 1–13. Tsapkina, E. N., Semenova, M. B., Pavlovskaya, G. E., & Tolstoguzov, V. B. (1992). The influence of incompatibility on the formation of adsorbing layers and dispersions of n-decane emulsion droplets in aqueous solution containing a mixture of 11S globulin from Vicia faba and dextran. Food Hydrocolloids, 6, 237–251. Tung, C. Y. M., & Dynes, P. J. (1982). elationship between viscoelastic properties and gelation in thermosetting systems. Journal Applied Polymer Science, 27, 569–580. Yilmazer, A. R., Carrillo, A. R., & Kokini, J. (1991). Effect of propylene glycol alginate and xanthenes gum on stability of o/w emulsions. Journal Food Science, 56, 513– 517.
Interactions of polysaccharides with b-lactoglobulin spread monolayers at the air – water interface R. Baezaa, C. Carrera Sanchezb, A.M.R. Pilosofa, J.M. Rodrı´guez Patinob,* a
Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria (1428), Buenos Aires, Argentina b Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, c/. Prof. Garcı´a Gonza´lez, 1. 41012, Sevilla, Spain Received 5 November 2003; revised 26 January 2004; accepted 5 March 2004
Abstract In the present work we have studied the static (film structure and elasticity) and dynamic characteristics (surface dilatational properties) of b-lactoglobulin (bLG) monolayers spread at the air –water interface in the presence of polysaccharides in the aqueous phase, at 20 8C and at pH 7. The measurements were performed on a fully automated Wilhelmy-type film balance. As polysaccharides with interfacial activity we have used propylene glycol alginates (PGA). To evaluate the effect of the degree of PGA esterification and viscosity, different commercial samples were studied-kelcoloid O (KO), kelcoloid LVF (KLVF) and manucol ester (MAN). Xanthan gum (XG) and l-carrageenan (lC) were studied as non-surface active polysaccharides. The results reveal a significant effect of surface active and non-surface active polysaccharides on static—when the polysaccharide was added in the subphase the p-A isotherms shifted to higher surface pressure values as the time increased-and dynamic—the presence of polysaccharide in the aqueous phase decreased the surface dilatational modulus of a pure b-lactoglobulin monolayer-characteristics of b-lactoglobulin monolayers. To explain the observed effects three phenomena were taken into account: (i) the ability of the polysaccharide to adsorb at the interface by it-self and to increase the surface pressure, (ii) the interfacial complexation of the polysaccharide with the adsorbed protein and (iii) the existence of a limited thermodynamic compatibility between the protein and polysaccharide, depending on the protein-polysaccharide system. q 2004 Elsevier Ltd. All rights reserved. Keywords: Protein; Polysaccharide; Gums; Hydrocolloid; Air–water interface; Monolayer; Surface tension; Interfacial rheology
1. Introduction Proteins and polysaccharides are present in many kinds of foods (Dickinson, 1992). The main role of proteins in foamed products is to stabilise the air –water interface through their capacity to lower the surface tension of water (Damodaran & Paraf, 1997). Polysaccharides are widely used for stabilisation of food emulsions and foams (Stephen, 1995; Renard, Della Valle, & Popineau, 2002). Most highmolecular-weight polysaccharides, being hydrophilic, do not have much of a tendency to adsorb at the air– water interface, but they can strongly enhance the stability of protein foams by acting as thickening or gelling agents (Dickinson, 2003). Alongside the use of non-surface-active polysaccharides in food foams as thickeners, there is some evidence that supports an additional role with some polysaccharides at the interfacial film. The formation of * Corresponding author. Tel.: þ 349-5-4556446; fax: þ349-5-455-7134. E-mail address: [email protected] (J.M. Rodrı´guez Patino). 0268-005X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2004.03.006
protein – polysaccharide complexes has been related to enhance the functionality of proteins adsorbed at fluid interfaces (Dickinson, 2003). Protein – polysaccharide interactions are sensitive to details of protein and polysaccharide structures as well as to pH (Dickinson, 2003; Tolstoguzov, 1997). Aqueous solutions of proteins and polysaccharides can exhibit one of the following phenomena: complex coacervation (complexation), miscibility and segregation. Complex coacervation occurs below the protein isoelectric point as a result of net electrostatic interactions between the biopolymers carrying opposite charges and implies the separation of two phases, one rich in the complexed biopolymers and the other phase depleted in both. Above the isoelectric point of the protein thermodynamic incompatibility between the protein and polysaccharide generally occurs because of the repulsive electrostatic interactions and different affinities towards the solvent (Tolstoguzov, 1997). Therefore, protein and polysaccharide
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may co-exist in a single phase (miscibility) in domains in which they mutually exclude one another or, above a critical concentration, segregate into different phases. Excluded volume effects can have the following manifestations: enhancement of the association of macromolecules, reduction in the critical concentration for gelation and increase in the rate of gelation (Capron, Nicolai, & Durand, 1999; Baeza, & Pilosof, 2001), enhancement of protein adsorption at fluid interfaces (Tsapkina, Semenova, Pavlovskaya, & Tolstoguzov, 1992). In the present work we have studied the static and dynamic characteristics of b-lactoglobulin (bLG) monolayers spread at the air –water interface in the presence of polysaccharides in the aqueous phase, at 20 8C and at pH 7. As polysaccharides with interfacial activity we have used propylene glycol alginates (PGA). To evaluate the effect of the degree of PGA esterification and viscosity, different commercial samples were studied. Xanthan gum (XG) and l-carrageenan (lC) were studied as non-surface active polysaccharide. Sulphated polysaccharides like carrageenans can interact with the positively charged groups in a protein more strongly than carboxylated hydrocolloids like XG at pH above the protein isoelectric point (Dickinson, 2003). Rheological properties of XG, along with its pH and temperature stability make it an ideal thickener for food emulsions and foams. XG is an anionic polysaccharide that produces high viscosities at low concentrations (Imeson, 1992). Enhanced hydration is attributed to side chains of anionic character consisting in units of a-D mannnose, b-D glucuronic and pyruvate, occurring on the cellulose structure backbone of xanthan. Xanthan, being highly hydrophilic and without any significant hydrophobic bonds, is not adsorbed at the air– water interface (Yilmazer, Carrillo, & Kokini, 1991). Nevertheless, xanthan promoted soy protein subunits aggregation at the air –water interface in foams based on native soy proteins (Carp, Bartholomai, & Pilosof, 1999). Those specific effects further influenced the foam stability of the mixed systems (Carp, Bartholomai, Relkin, & Pilosof, 2001). One possible interpretation of these findings is that the gum adsorbs onto the protein, forming a combined structure of a primary protein layer predominantly in contact with the air phase. Following this last hypothesis, the surface tension might be dominated by the primary protein layer, covered by and adsorbed polysaccharide layer in strong electrostatic contact, dominating the surface rheology (Galazka & Dickinson, 1995). One distinctly group of surface-active polysaccharides are the propylene glycol esters of alginic acid (PGA), a high molecular weight linear polysaccharides composed of 1,4 linked- D -mannuronic acid and L -guluronic acid (Sarker & Wilde, 1999). There are produced with a range of viscosities and degrees of esterification. The increasing of the degree of esterification reduces the overall hydrophilic character of the molecules and imparts surface-active properties. The resulting ability of PGA to reduce the surface tension of
water as well as increase the viscosity of the water phase make them suitable as stabilisers and foaming agents. Substitution of more than 50% of the carboxyl groups also produces greater acid and calcium ion tolerance than other alginates. Propylene glycol alginate is an example of a polysaccharide used in food foams, such as beer, to aid ‘head’ retention of the foam (Sarker & Wilde, 1999). Due to its surface-active character, competitive adsorption of PGA could occur in mixtures of this polysaccharide and proteins. In addition, formation of protein –polysaccharide complexes at the interface could also occur. The interaction between PGA and milk proteins in foams was studied by Ahmed and Dickinson (1991). The foaming properties were strengthened at pH value above 7, where both biopolymers carried negative charges, revealing the possibility of formation of electrostatic complexes between positively charged patches in the proteins and carboxyl groups in PGA. A more quantitative indication of non-covalent complex formation between PGA and caseinate, whey protein isolates and a-lactalbumin at the oil– water interface was provided by film surface viscosity (Dickinson & Euston, 1991). More recently, the foam stability of a mixed bovine serum albumin/Tween 20 solution at pH 4.2 was found to be significantly increased in the presence of PGA (Sarker & Wilde, 1999). The principal mechanism is thought to be non-covalent cross-linking of the protein molecules at the surface. b-lactoglobulin (bLG), the most abundant protein in whey, is a globular protein of molecular mass 18.3 kDa, stabilised by two internal disulfide cross-linking, that exhibits good foaming properties (Tornberg, 1979). The structural and dynamic properties of b-lactoglobulin at the air –water interface, have been extensively studied in the past years (Horne & Rodrı´guez Patino, 2003; Rodrı´guez Nin˜o, Carrera, Cejudo, & Rodrı´guez Patino, 2001, Rodrı´guez Patino, Carrera, Rodrı´guez Nin˜o, & Cejudo, 2001) so that this protein is a good model to study the interactions of non-surface active and surface active polysaccharides with proteins at the air –water interface.
2. Materials and methods 2.1. Materials b-lactoglobulin (bLG) was supplied by Danisco Ingredients (Denmark). The powder composition was: protein 92%, b-lactoglobulin . 95%, a-lactalbumin , 5%. The polysaccharides l-carrageenan (lC) and xantham gum (XG) were provided by BIOTEC (Argentina) and propylene glycol alginates (PGA) were from ISP Alginates Inc. (USA). The PGA used were: kelcoloid O (KO), kelcoloid LVF (KLVF) and manucol ester (MAN). The degree of esterification and viscosity of propylene glycol alginates are shown in Table 1. The surface pressure of aqueous solutions of polysaccharides at 0.1 and 0.5 wt%—which
R. Baeza et al. / Food Hydrocolloids 18 (2004) 959–966 Table 1 Degree of esterification and viscosity of propylene glycol alginates PGA
Degree of esterification
Viscositya
Manucol ester (MAN) Kelcoloid LVF (KLVF) Kelcoloid O (KO)
High Medium High
High (11.8 cps) High (13.9 cps) Low (4.7 cps)
a
viscosity (60 s21) of 0.5% wt/wt solution.
was determined using an automatic drop tensiometer as described elsewhere (Rodrı´guez Patino, Rodrı´guez Nin˜o, & Carrera, 1999)—are shown in Table 2. It can be seen that all the PGA used in this study have a significant surface activity denoting the surface-active character of these polysaccharides. The surface pressure of XG is practically zero denoting the non-surface active characteristic of this polysaccharide. However, the surface pressure of l-carrageenan is different to zero denoting the presence of some impurities in the commercial sample. Thus, a purification of this polysaccharide was necessary before the experiments in the surface film balance, by the continuous suction of the adsorbed impurities until the surface pressure of the aqueous solution turns to zero.
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complete spreading, adsorption and rearrangements of the protein, 30 min was allowed to elapse before measurements were taken. In the systems with surface-active polysaccharides in the aqueous phase the protein was spread at the interface using pure water as sub-phase. Then a concentrated PGA solution was added to the sub-phase with a syringe. The final concentration of polysaccharide in the sub-phase was approximately 0.05% wt/wt in. In the presence of nonsurface active polysaccharides (lC and XG), the protein was spread directly on the surface of polysaccharide solutions at 0.05% wt/wt. The sub-phase temperature was maintained at 20 8C by water circulation from a thermostat, within an error range of 0.5 8C. The reproducibility of the surface pressure results was better than ^ 0.4 mN/m. 2.3. Film elasticity The elasticity was calculated from the slope of the p-A isotherm as E ¼ 2Aðdp=dAÞT : The elasticity defined here is the elasticity at zero deformation rate. The film elasticity is a measure of the film resistance to a change in area and can be calculated directly from the slope of the p-A isotherm at constant temperature.
2.2. Surface film balance
2.4. Surface dilatational rheology
The measurements of the surface pressure ðpÞ vs. average area per molecule (A) were performed on fully automated Wilhelmy-type film balance (KSV 3000, Finland) as described elsewhere (Rodriguez Patino et al., 2001). The maximum area of the trough between the two oscillating barriers is 51.5 £ 15 cm2. To form the protein surface film, bLG was spread in the form of a solution, using Milli-Q ultrapure water at pH 7, adjusted by a commercial buffer solution (Sigma, . 99.5%) called trizma((CH2OH)3CNH2/ (CH2OH)3CNH3Cl). Ionic strength was 0.05 M in all the experiments. The sub-phase solutions consisted in 0.05% wt/ wt polysaccharide solutions prepared with Milli-Q water. Aliquots of 400 ml of aqueous solutions of bLG (1.6 1024 mg/ml) at pH 7 were spread on the interface using the method developed by Trurnit (1960). To allow
Determination of surface rheological parameters—such as surface dilatational modulus ðEÞ; its elastic and viscous components, and the loss angle tangent—were carried out in a home modified-Wilhelmy-type film balance as described elsewhere (Rodriguez Patino et al., 2001). In this method the surface was subjected to small periodic sinusoidal compression and expansion cycles by means of two oscillating barriers at a given frequency ðvÞ and amplitude ðDA=AÞ and the response of the surface pressure ðpÞ was monitored. The conditions used in this study were a frequency of 50 mHz and an amplitude of 5%. Surface pressure was directly measured by means of two roughened platinum plates situated on the surface between the two barriers. The surface dilatational modulus ðEÞ derived from the change in surface pressure resulting from a small change in surface area may be described (Lucassen and van den Tempel, 1972) by E ¼ 2dp=d ln A: The dilatational modulus is a complex quantity, composed of real (the surface dilatational elasticity, Ed ¼ lEl cos u) and imaginary (the surface dilatational viscosity, Ev ¼ lEl sin uÞ parts. The loss angle tangent tanu ¼ Ev =Ed is a measurement of the solid character of the film and is near zero for pure elastic films.
Table 2 Surface pressure of aqueous solutions of polysaccharides at 20 8C and at pH 7, after 3600 s of adsorption Concentration of polysaccharide in the aqueous solution
Manucol ester (MAN) Kelcoloid LVF (KLVF) Kelcoloid O (KO) Xanthan gum (XG) l-Carrageenan (lC)
0.1%wt
0.5%wt
6.8 6.3 9.3 <0 –
6.9 6.3 9.3 <0 7.8a
a The surface pressure of a l-Carrageenan solution at 0.5%wt was zero after the suction of the surface-active impurity.
3. Results 3.1. Monolayer structural characteristics The monolayer structural characteristics can be obtained through the p-A isotherm. The p-A isotherm for
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b-lactoglobulin spread on aqueous subphase at pH 7 is shown in Fig. 1. b-lactoglobulin monolayers have a liquidexpanded-like structure (Rodriguez Patino et al., 2001). It appears that a critical surface pressure and surface concentration exist at which the film properties change significantly. This critical surface pressure of 12 mN/m represents the transition between a monolayer with a more expanded structure (state 1) towards a monolayer in which the proteins form a more condensed structure (state 2). Nevertheless, according to Graham and Phillips (1979), b-lactoglobulin would retain elements of the native structure not fully unfolded at the interface, even in the more expanded configuration (Rodriguez Patino et al., 2001). When the surface active PGA were added in the subphase at a concentration of 0.05% wt/wt, the p-A isotherms shifted to higher surface pressure values as the time increased, due to the adsorption and penetration of the polysaccharide into the protein monolayer (Figs. 1– 3). The reproducibility of the isotherms after continuous compression –expansion cycles was reached after 18 – 22 h, indicating that the adsorption and penetration of PGA into the protein monolayer and its subsequent rearrangement is a slow process. In the presence of non-surface active polysaccharides different results were obtained. The presence of XG strongly shifted the p-A isotherms to higher surface pressure values as the time increased (Fig. 4), but l-carrageenan induced only small changes in the p-A isotherm (data not shown). The p-A isotherms at the maximum adsorption time (the pseudo steady state p-A isotherm) of b-lactoglobulin and b-lactoglobulin þ polysaccharide mixed systems are shown in Fig. 5. It can be seen that the p-A isotherms for b-lactoglobulin þ PGA and b-lactoglobulin þ XG were separated from the surface pressure axis indicating an apparent less condensed monolayer structure because in the molecular area we assume that only bLG molecules are
Fig. 1. p-A isotherms of b-lactoglobulin monolayers spread on aqueous solutions of Kelcoloid O (KO) at 20 8C and at pH 7. Key: (1, —) blactoglobulin spread on pure water and after the addition of Kelcoloid O to the aqueous phase and after a waiting time of (2, pppp) 16 h and (3, ---) 18 h.
Fig. 2. p-A isotherms of b-lactoglobulin monolayers spread on aqueous solutions of Kelcoloid LVF (KLVF) at 20 8C and at pH 7. Key: (1, —) b-lactoglobulin spread on pure water and after the addition of Kelcoloid LVF to the aqueous phase and after a waiting time of (2, – –) 4 h, (3, · · ·) 18 h, and (4, -·-) 22 h.
spread on the interface. Among the different PGA, MAN showed the highest surface activity as it induced the highest surface pressure increase. KLVF and KO induced a much lower increase in surface pressure. Differences among them were only apparent at low molecular areas where KLVF exhibited slightly higher surface activity. In spite of the lack of a surface activity by it-self, xanthan showed a behaviour similar to PGA. Moreover, at low molecular areas XG induced higher surface pressure than KLVF and KO. The l-carrageenan slightly increased the surface pressure at low molecular areas. By plotting the elasticity versus surface pressure for pure bLG and mixed systems (Fig. 6) a more clear evidence of the changes in the monolayer structure may be obtained. The elasticity of pure bLG monolayer increased with the surface pressure, reached a maximum as the change
Fig. 3. p-A isotherms of b-lactoglobulin monolayers spread on aqueous solutions of Manucol ester (MAN) at 20 8C and at pH 7. Key: (1, —) b-lactoglobulin spread on pure water and after the addition of MAN to the aqueous phase and after a waiting time of (2, – –) 1 h, (3, · · ·) 18 h, and (4, -·-) 22 h.
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Fig. 4. p-A isotherms of b-lactoglobulin monolayers spread on aqueous solutions of xanthan gum (XG) at 20 8C and at pH 7. Key: (1, —) b-lactoglobulin spread on pure water and after the addition of xanthan gum to the aqueous phase and after a waiting time of (2, – –) 1.5 h, (3, · · ·) 13 h, and (4, -·-) 14 h.
between states 1 and 2 takes place and then decreased at the higher surface pressures as the monolayer collapses and the formation of a multilayer takes place (Rodriguez Patino et al., 2001). Fig. 6 shows that the influence of the polysaccharide prevails at low surface pressures (at p , 12 mN/m) as bLG adopts the state less condensed (state 1). In this region the elasticity of the film was reduced up to 50% by the presence of MAN, KO and KLVF (Fig. 6a). This effect may be due to the penetration of polysaccharides in the protein film, which hinder the interactions within the protein aminoacid residues at the air –water interface. The non-surface active XG and lC also reduced the elasticity of pure bLG monolayer (Fig. 6b) but in a lower extent than surface active polysaccharides. This effect may be due to the interactions between protein and polysaccharide that affect the original structure of the protein monolayer. These results
Fig. 6. Surface pressure dependence of the elasticity of (—) b-lactoglobulin monolayers spread on water and after the addition of polysaccharides to the aqueous phase. (a) Surface active polysaccharide: (K) Kelcoloid O, (q) Kelkoid KLF, and (W) Manucol ester. (b) Non-surface active polysaccharide: (W) l-carrageenan, and (S) xanthan gum. Temperature 20 8C. pH 7.
would also suggest that the protein in the presence of these polysaccharides would be forced to adopt the more condensed state (state 2) even at low surface pressures. In the region where the protein adopts the state 2 (at 12 , p , 25 mN/m), the elasticity decreased and lower differences were observed among the systems (the elasticity varied within 20 and 30 mN/m). Above a surface pressure of 25 mN/m, in the monolayer collapse region, the elasticity tended to decrease no matter what the polysaccharide. 3.2. Surface dilatational characteristics
Fig. 5. Pseudo steady state p-A isotherms of b-lactoglobulin monolayers spread on aqueous solutions of polysaccharides at 20 8C and at pH 7. Key: (1, —) b-lactoglobulin spread on pure water and after the addition of (2, ----) Kelcoloid O, (3, · · ·) Kelkoid KLF, (4, -·-) Manucol ester, (5, -··-) l-carrageenan, and (6,þþ þ ) xanthan gum to the aqueous phase.
The values of surface dilatational modulus (data not shown) were very similar to that of the dilatational elasticity supporting the elastic character of the film. The dilatational elasticity ðEd Þ of bLG monolayer behaved as previously reported (Rodriguez Patino et al., 2001). The dilatational elasticity increased with surface pressure up to a maximum close to the surface pressure at which the monolayer collapses (Fig. 7). The presence of lC in the aqueous
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Fig. 7. Surface pressure dependence of the surface dilatational elasticity (Ed) of (A) b-lactoglobulin monolayers spread on water and after the addition of polysaccharides to the aqueous phase. Surface active polysaccharide: (K) Kelcoloid O, (L) Kelkoid KLF, and (W) Manucol ester. Non-surface active polysaccharide: (X) l-carrageenan, and (V) xanthan gum. Temperature 20 8C. pH 7.
subphase slightly modified the dilatational elasticity of the monolayer. A small decrease in Ed was observed at low surface pressure and at the collapse point. The PGA strongly decreased Ed at all surface pressures, being MAN the PGA that more decreased the solid character of the film, especially at the higher surface pressures. XG showed a very strong effect as it produced the largest reduction in Ed ; mainly at surface concentrations below 20 mN/m. b-Lactoglobulin monolayer behave, from a rheological point of view, as viscoelastic, with a loss angle tangent ðtan uÞ within 0.05 –0.2 (Fig. 8). These low values point out the like-gel character of the monolayers (Tung & Dynes, 1982). tan u decreased with increasing surface pressure in agreement with the existence of higher protein– protein
Fig. 8. Surface pressure dependence of the loss angle tangent (tan u) of (A) b-lactoglobulin monolayers spread on water and after the addition of polysaccharides to the aqueous phase. Surface active polysaccharide: (K) Kelcoloid O, (L) Kelkoid KLF, and (O) Manucol ester. Non-surface active polysaccharide: (X) l-carrageenan, and (V) xanthan gum. Temperature 20 8C. pH 7.
interactions. Except XG at lower surface pressures, the presence of polysaccharide decreased the viscoelasticity of bLG monolayer. Among PGA, KO less affected the viscoelastic character of the monolayer and MAN and KLVF showed a similar behaviour. The non-surface active polysaccharides showed opposite effects on the viscoelastic character of the film. At low surface pressures (in the region where the protein adopts the state 1), lC strongly decreased the viscoelasticity but the opposite was observed with XG. In fact, in the presence of lC the viscous component of the dilatational modulus of the mixed system increased in comparison to the protein alone. However, the presence of xanthan greatly decreased the viscous component of the dilatational modulus resulting in the increase of the viscoelastic character of the film.
4. Discussion The results of static (film structure) and dynamic (surface dilatational properties) of b-lactoglobulin and polysaccharide mixed systems reveal a significant effect of polysaccharide, even of the non-surface active xanthan and lC, on bLG monolayer. To explain the observed effects three phenomena must be taken into account: (i) the ability of the polysaccharide to adsorb at the interface by itself and to increase the surface pressure, (ii) the interfacial complexation of the polysaccharide with the adsorbed protein and (iii) the existence of a limited thermodynamic compatibility between the protein and polysaccharide. Anchorage of the polysaccharide at the interfacial film may occur by mechanism (i) or (ii), depending on the chemical structure of the polysaccharide and on the pH. Once the polysaccharide is into or attached by complexation to the interface, exclusion volume effects between both biopolymers at neutral pH could lead to a rise of chemical potential or, in other words, to a modification of the thermodynamic activity of the protein at the interface (Pavlovskaya, Semenova, Thzapkina, & Tolstoguzov, 1993). Therefore, the protein at the air – water interface would perform as a more concentrate film (i.e. with a more condensed structure), leading to an increase in the surface pressure, according to the results of Figs. 1– 3 and 5. It has been demonstrated that xanthan addition to soy protein solutions at neutral pH had an effect similar to that observed by increasing protein concentration arising mainly from excluded volume effects (Carp et al., 1999). The implication of some protein patches in electrostatic interactions with XG or lC or the penetration of PGA into the interface would interfere with the interactions between bLG molecules resulting in a decreasing in the surface dilatational elasticity (Fig. 7). The decreased elasticity at p , 12 mN/m, as bLG adopts the state 1 (Fig. 6), and the decreased surface dilatational elasticity at every surface pressures (Fig. 7) of bLG monolayers in the presence of polysaccharide reveals a decreasing in the degree of protein association at
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the interface, as well as the increased fluidification of the film (as observed by the increased tan u in Fig. 8, which is consistent with the existence of lower protein– protein interactions in the presence of polysaccharides. b-Lactoglobulin spread films on aqueous solutions of non-surface active polysaccharides. The adsorption of pure xanthan (XG) and l-carrageenan (lG) at the air– water interface is unlikely. Xanthan did not cause a decrease in surface tension (Carp et al., 2001), which was consistent with previous reports (Yilmazer et al., 1991) and purified lC behaved similarly (Table 2). Thus, the modification of surface pressure (Figs. 4 and 5) and rheological properties (Figs. 6b, 7, and 8) of the protein monolayer in the presence of non-surface active polysaccharide in the aqueous phase necessary suggests the participation of XG and lC at the interface by the mechanism (ii). At the neutral pH used in this study, a protein – polysaccharide repulsive interaction may be expected. b-Lactoglobulin is negatively charged and XG is a nonionic polysaccharide with side chains of anionic character consisting in units of a-D mannose, b-D glucuronic and pyruvate, occurring on the cellulose structure backbone. On the other hand, l-carrageenan is a highly sulphated polysaccharide, negatively charged at neutral pH. In bulk solution, the mixtures of whey proteins and XG or lC at pH 7 appeared to be governed by segregative or limited thermodynamic compatibility phenomena (Sanchez, Schmitt, Babak, & Hardy, 1997; Baeza & Pilosof, 2001). However, local net attractive interactions between proteins and polysaccharides may also occur. Under the adsorption of the protein at the interface the character of protein – polysaccharide interactions may be different than in bulk solution because of the altered conformation of protein at the interface. One of the most important properties that differentiate carrageenan from other hydrocolloids is its ability to complex or interact with milk proteins. Sulphated polysaccharides form fairly strong reversible complexes with proteins, even at neutral pH (Dickinson, 2003). Interestingly, lC is the polysaccharide that has a lower effect on bLG monolayer structure (Fig. 5) and surface dilatational elasticity (Fig. 7), but conversely produces a higher fluidification in the film (Fig. 8). Clearly, the interfacial complexation of polysaccharide with adsorbed protein and/ or the repulsive interactions between both biopolymers, according to mechanism (ii), may produce complex effects on the interfacial characteristics (both static and dynamic) of the mixed films. Evidence for the synergistic interaction of xanthan and soy proteins at the air– water interface was previously provided by measurements of surface shear viscosity (Carp, Elizalde, Bartholomai, & Pilosof, 1997) and electrophoresis analysis of protein aggregation at the air – water interface (Carp et al., 1999). One possible interpretation of these findings is that the gum adsorbs onto the protein, forming a combined structure with a primary protein layer
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predominantly in contact with the air phase. Following this last hypothesis, the surface pressure might be dominated by the primary protein layer and should not be changed by the polysaccharide that would mainly dominates the surface rheology (Fig. 7). However, the strong increase of surface pressure observed in bLG monolayer in the presence of xanthan (Figs. 4 and 5) may be taken as an evidence of the ‘concentrating effect’ (condensation of the bLG monolayer structure) arising from the limited thermodynamic compatibility between the biopolymers, according to the mechanism (iii). b-Lactoglobulin spread films on aqueous solutions of surface active polysaccharides. As a result of the ester group, propylene glycol alginates (PGA) reduce the surface tension of water, a phenomenon, which imparts a surface active character to these polysaccharides. The penetration of PGA at the interface, according to mechanism (i) may reduce the protein– protein interactions, which explains the lower values of the surface dilatational elasticity (Fig. 7). An increasing in the degree of esterification reduces the overall hydrophilic character of the molecule and imparts higher surface active properties to the polysaccharide aqueous solution. PGA also differ in their viscosities, and thus in their molecular weights (Imeson, 1992). The surface activity of PGA (in the absence of the protein) followed the order KO . MAN . KLVF (Table 2). The highest surface activity of KO is consistent with the high degree of esterification and low viscosity (i.e. with the lower molecular weight) of this PGA. On the other hand, KLVF because of the lowest degree of esterification and higher viscosity showed the worst performance. Nevertheless, when PGA is adsorbed onto the bLG film, the order of effectiveness in increasing surface pressure (Figs. 1– 3 and 5) was MAN . KLVF . KO. This result cannot be only attributed to the high degree of esterification and necessarily points out the role of the viscosity (i.e. the molecular weight) of PGA, which is indicative of the degree of incompatibility with the protein. The increasing of the molecular weight of the PGA is expected to reduce the biopolymer compatibility (Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998). According to this, the different effects of PGA on p-A isotherms (Fig. 5) may be explained as follows. The greatest increase of surface pressure caused by MAN (Figs. 3 and 5) should be attributed to its high degree of esterification, which allows it to penetrate into the interface. Nevertheless, the possibility of interfacial complexing must not be discarded. Ahmed and Dickinson (1991) suggested the possibility of formation of electrostatic complexes between PGA and milk proteins in foams at pH 7. In addition, due to its high viscosity/molecular weight they would force the protein to concentrate at the interface due to exclusion volume effects. Even if KO had a degree of esterification similar to MAN, due to its low viscosity, would contribute to a lesser extent to concentrate the protein at the interface.
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Finally, the fact that all the polysaccharides, being surface-active or not, caused the transition towards state 2 to occur at lower surface areas is an evidence of the existence of a volume exclusion effects at the interface. Otherwise, the non-surface active polysaccharides would only modify the rheology of the protein film without affecting the surface pressure.
Acknowledgements This research was supported by CYTED through project XI.17 and CICYT through grant AGL2001-3843-C02-01. The authors also acknowledge the support from Universidad de Buenos Aires and Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas de la Repu´blica Argentina.
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